Project Summary/Abstract This project designs, develops, and automates a novel system to process solid tissue into single-cells, single-nuclei, and high molecular weight DNA using disposable cartridges without user intervention. The system will support downstream single-cell RNA or DNA sequencing, single-nuclei RNA or DNA sequencing, and long read sequencers and optical mapping. The long-term goal of the project is to enable researchers and clinicians with sample-to-answer solutions for fully integrated sequencing of solid tissues including biopsy samples. In the Phase I project, automated workflows are developed and optimized in novel, fully integrated cartridge designs. The prototype system is first benchmarked against an S2 breadboard system and manual processes for single-cell production from mouse tissues. Cartridges, mechanical disruption, enzymology, and workflows are further developed and optimized for mouse and rat tissues for fully integrated processing performance. A cartridge for 15 mg tissue specimens is designed and tested. Processing single-cells into high molecular weight DNA is tested using the magnetic separations in the cartridge and on other devices. The automated preparation of nuclei will be tested and developed on prototype systems. Single-cell suspensions prepared on the prototype will be processed into nuclei by chemical lysis in the cartridge and magnetic purification of nuclei with paramagnetic beads tested. Enzymatic formulations with gentler processing will be studied. Workflows and cartridges to directly process tissue into nuclei will be developed and tested. The single-sample prototype systems will be tested externally at the J Craig Venter Institute and the Broad Institute. Phase I testing will design the architecture of a multi-sample pre- production prototype system with additional capabilities to be built and tested in Phase II. An optical module will be added to determine viability and titer on the system and potentially allow the system to adjust dissociation protocols ?on-the-fly.? Integrated magnetic processing will be further developed to purify cell-types, nuclei and other organelles, and DNA. The project output will be 1) a validated single-sample tissue-to-cell, nuclei, or high molecular weight DNA automated prototype, and 2) a multiple-sample design architected for Phase II development.